Team:Evry/Interlab Study/08-13-2014

From 2014.igem.org

Contructions PSB1C3 with I20260, J23101-E1010 and K823012-E1010

PCR products were cleaned with the GeneJET purification kit (Thermo Scientific)followed by DNA quantification with the NanoDrop 2000 (Thermo Scientific).
Purified PCR product of BBa_J61002/J23101 and BBa_J61002/K823012 concentration were respectively: 53.9 ng/µl and 54.1 ng/µl.

Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders. Gel running 45 minutes at 100 mV in TAE 1X buffer.

Figure 2: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 1 and 7: Purple 2-Log ladder NEB, Lane 2: BBa_J23101 purified PCR product, Lane 3: BBa_K823012 purified PCR product, Lane 4: BBa_E0240 purified PCR product, Lane 5: BBa_I20260 purified PCR product and Lane 6: pBHR1 purified PCR product

Profiles were same as before PCR clean up. To sequence the amplified parts, we decided to a purification of fragments from the gel as presented below.

Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. Microwave 30s by 30s until agarose total dissolution. Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye, at 10µl of the mix per well. Gel running 45 minutes at 100 mV in TAE 1X buffer.

Figure 3: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 4 and 8: Purple 2-Log ladder NEB, Lane 1, 2 and 3: BBa_E0240 purified PCR product, Lane 5, 6 and 7: BBa_I20260 purified PCR product

Four pieces of gel were sampled in four tubes to perform a DNA purification from gel.

Aug 13